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ApoTox-Glo™ Triplex Assay

The ApoTox-Glo™ Triplex Assay combines three assay chemistries to easily assess viability, cytotoxicity and apoptosis events in the same cell-based assay well. First, viability and cytotoxicity are determined by measuring two differential protease biomarkers simultaneously with the addition of a single nonlytic reagent containing two peptide substrates. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant peptide substrate (GF-AFC Substrate). The substrate enters intact cells, where it is cleaved to generate a fluorescent signal proportional to t...

The ApoTox-Glo™ Triplex Assay combines three assay chemistries to easily assess viability, cytotoxicity and apoptosis events in the same cell-based assay well. First, viability and cytotoxicity are determined by measuring two differential protease biomarkers simultaneously with the addition of a single nonlytic reagent containing two peptide substrates. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant peptide substrate (GF-AFC Substrate). The substrate enters intact cells, where it is cleaved to generate a fluorescent signal proportional to the number of living cells. This live-cell protease activity marker becomes inactive upon loss of membrane integrity and leakage into the surrounding culture medium. A second, cell-impermeant, fluorogenic peptide substrate (bis-AAF-R110 Substrate) is used simultaneously to measure dead-cell protease activity that has been released from cells that have lost membrane integrity. This results in ratiometric, inversely correlated measures of cell viability and cytotoxicity. The ratio of viable cells to dead cells is independent of cell number and, therefore, can be used to normalize data. A second reagent containing luminogenic DEVD-peptide substrate for caspase-3/7 and Ultra-Glo™ Recombinant Thermostable Luciferase is added. Caspase-3/7 cleavage of the substrate releases luciferin, which is a substrate for luciferase and generates light. The light output, measured with a luminometer, correlates with caspase-3/7 activation as a key indicator of apoptosis.

Features - Benefits

Measure Viability, Cytotoxicity and Apoptosis in the Same Sample Well: Determine mechanism of cell death for cells in the same sample well.

Normalize Data with a Built-In Control: The ratio of the number of live cells/number of dead cells is independent of cell number and normalizes data. This normalization makes results more comparable well-to-well, plate-to-plate and day-to-day.

Easily Automate this Flexible Assay: Component volumes can be scaled to meet throughput needs. Amenable to automation in 96- and 384-well plates.

Improve Efficiency and Save Lab Budget: Reduce cell culture and labor costs by performing three assays in a single well.

Applications

Determine mechanism of cell death.

Profile compound libraries for cytotoxic risk.

Notes

Cat.# G6320 contains sufficient reagents for 100 assays in a 96-well plate format or 400 assays in a 384-well format. Cat.# G6321 contains sufficient reagents for 500 assays in a 96-well plate format or 2,000 assays in a 384-well format.

References

Niles, A.L. et al. (2007) A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal. Biochem.366, 197–206.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

ApoTox-Glo™ Triplex Assay with suspension Jurkat cells treated with staurosporine. Staurosporine treatment for 6 hours results in a dose-dependent decrease in viability and increase in cytotoxicity with an increase in caspase-3/7 activity consistent with apoptosis.

Camptothecin treatment of Jurkat cells for 48 hours results in dose-dependent decrease in viability, no cytotoxicity and increase in caspase-3/7 activity, consistent with cell-cycle arrest and early-phase apoptosis.

Camptothecin treatment of Jurkat cells for 48 hours results in dose-dependent decrease in viability, no cytotoxicity and increase in caspase-3/7 activity, consistent with cell-cycle arrest and early-phase apoptosis.

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